Production Of Doubled Haploid Plants Research Articles

Trichostatin A Affects Developmental Reprogramming of Bread Wheat Microspores towards an Embryogenic Route.

Microspores can be developmentally reprogrammed by the application of different stress treatments to initiate an embryogenic pathway leading to the production of doubled haploid (DH) plants. Epigenetic modifications are involved in cell reprogramming and totipotency in response to stress. To increase microspore embryogenesis (ME) efficiency in bread wheat, the effect of the histone deacetylase inhibitor trichostatin A (TSA) has been examined in two cultivars of wheat with different microspore embryogenesis response. Diverse strategies were assayed using 0–0.4 µM TSA as a single induction treatment and after or simultaneously with cold or mannitol stresses. The highest efficiency was achieved when 0.4 µM TSA was applied to anthers for 5 days simultaneously with a 0.7 M mannitol treatment, producing a four times greater number of green DH plants than mannitol. Ultrastructural studies by transmission electron microscopy indicated that mannitol with TSA and mannitol treatments induced similar morphological changes in early stages of microspore reprogramming, although TSA increased the number of microspores with ’star-like’ morphology and symmetric divisions. The effect of TSA on the transcript level of four ME marker genes indicated that the early signaling pathways in ME, involving the TaTDP1 and TAA1b genes, may be mediated by changes in acetylation patterns of histones and/or other proteins.

Meiotic crossover reduction by virus-induced gene silencing enables the efficient generation of chromosome substitution lines and reverse breeding in Arabidopsis thaliana.

Plant breeding applications exploiting meiotic mutant phenotypes (like the increase or decrease of crossover (CO) recombination) have been proposed over the last years. As recessive meiotic mutations in breeding lines may affect fertility or have other pleiotropic effects, transient silencing techniques may be preferred. Reverse breeding is a breeding technique that would benefit from the transient downregulation of CO formation. The technique is essentially the opposite of plant hybridization: a method to extract parental lines from a hybrid. The method can also be used to efficiently generate chromosome substitution lines (CSLs). For successful reverse breeding, the two homologous chromosome sets of a heterozygous plant must be divided over two haploid complements, which can be achieved by the suppression of meiotic CO recombination and the subsequent production of doubled haploid plants. Here we show the feasibility of transiently reducing CO formation using virus-induced gene silencing (VIGS) by targeting the meiotic gene MSH5 in a wild-type heterozygote of Arabidopsis thaliana. The application of VIGS (rather than using lengthy stable transformation) generates transgene-free offspring with the desired genetic composition: we obtained parental lines from a wild-type heterozygous F1 in two generations. In addition, we obtained 20 (of the 32 possible) CSLs in one experiment. Our results demonstrate that meiosis can be modulated at will in A.thaliana to generate CSLs and parental lines rapidly for hybrid breeding. Furthermore, we illustrate how the modification of meiosis using VIGS can open routes to develop efficient plant breeding strategies.

Suppression of Metacaspase- and Autophagy-Dependent Cell Death Improves Stress-Induced Microspore Embryogenesis in Brassica napus.

Microspore embryogenesis is a biotechnological process that allows us to rapidly obtain doubled-haploid plants for breeding programs. The process is initiated by the application of stress treatment, which reprograms microspores to embark on embryonic development. Typically, a part of the microspores undergoes cell death that reduces the efficiency of the process. Metacaspases (MCAs), a phylogenetically broad group of cysteine proteases, and autophagy, the major catabolic process in eukaryotes, are critical regulators of the balance between cell death and survival in various organisms. In this study, we analyzed the role of MCAs and autophagy in cell death during stress-induced microspore embryogenesis in Brassica napus. We demonstrate that this cell death is accompanied by the transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), an increase in MCA proteolytic activity and the activation of autophagy. Accordingly, inhibition of autophagy and MCA activity, either individually or in combination, suppressed cell death and increased the number of proembryos, indicating that both components play a pro-cell death role and account for decreased efficiency of early embryonic development. Therefore, MCAs and/or autophagy can be used as new biotechnological targets to improve in vitro embryogenesis in Brassica species and doubled-haploid plant production in crop breeding and propagation programs.

Production of doubled haploid plants ofCucurbitaceaefamily crops through unpollinated ovule culture in vitro

Production of doubled haploid plants of<i>Cucurbitaceae</i>family crops through unpollinated ovule culture in vitro

An Overview of Doubled Haploid Plant Production in Cucurbita Species

F1 cultivars are currently used in vegetable production because of their many superior agronomic advantages (high yield and quality, resistance/tolerance to biotic and/or abiotic stress factors, wide adaptability, etc.). It is not possible to produce hybrid vegetables without homozygous pure parental lines with defined traits and high combining ability. However, the production of homozygous pure parents needs a long time, great efforts, and high production cost. Moreover obtaining an F1 hybrid variety takes 8-10 years with traditional breeding methods, especially in a highly open-pollinated species such as Cucurbita spp. Doubled haploid (DH) technologies (parthenogenesis, androgenesis, and gynogenesis), called biotechnological breeding methods, have become an alternative to classical breeding methods and provide to obtain pure lines within 1-2 years by adapting to these technologies in F1 hybrid breeding programs. Although the DH technologies considered a meaningful approach to enhance the production of valuable double haploid plants, the implementation of these techniques needs labor-intensive efforts, high experiences, and advanced technologies. The main objective of this review to provide a summary of DH techniques applied in the Cucurbita species in the current progress.

Chromosome doubling methods in doubled haploid and haploid inducer-mediated genome-editing systems in major crops.

The doubled haploid technique aims to generate pure inbred lines for basic research and as commercial cultivars. The doubled haploid technique first generates haploid plants and is followed by chromosome doubling, which can be separated in time or overlapped, depending the procedure for each species. For a long time, much effort has been focused on haploid production via androgenesis, gynogenesis, or parthenogenesis. The obtention of haploid plants has frequently required more optimization and has lagged behind research and improvements in chromosome doubling methods. Nevertheless, chromosome doubling has recently been of renewed interest to increase the rates and efficiency of doubled haploid plant production through trialing and optimizing of different procedures. New antimitotic compounds and application methods are being studied to ensure the success of chromosome doubling once haploid material has been regenerated. Moreover, a haploid inducer-mediated CRISPR/Cas9 genome-editing system is a breakthrough method in the production of haploid plant material and could be of great importance for species where traditional haploid regeneration methods have not been successful, or for recalcitrant species. In all cases, the new deployment of this system will demand a suitable chromosome doubling protocol. In this review, we explore the existing doubled haploid and chromosome doubling methods to identify opportunities to enhance the breeding process in major crops.

Comparison of Orchard-grass and Sweet Maize for Doubled Haploid Plant Production via Wide Hybridization in Bread Wheat

In this study, the potential of haploid regeneration was investigated in hybridization of six bread wheat F1 hybrids known response to another culture with orchard-grass (Dactylis glomerata L.) and sweet maize varieties (Baron, Challenger and Merit). A total of 150 wheat spikes were pollinated with orchard grass and sweet maize and 2730 pseudo-seeds were produced. Although the high rate of developed pseudo-seeds was developed from bread wheat F1 hybrids × orchard-grass, no embryos were produced. Developed pseudo-seeds (2057 number) of bread wheat × sweet maize produced 53 haploid embryos and only 8 of them were regenerated. Developed green plantlets were vernalized and applied colchicine and only four of them produced fertile seeds. The highest rate (5.9) of haploid embryo formation within wheat genotypes was determined in DH20 × Kate A-1. Although the highest haploid embryo formation was observed in Challenger with 3.5% among sweet maize genotypes, it had no effect on plant regeneration. Also, the mixture of pollen of sweet maize varieties increased haploid plant regeneration. It has been observed that some F1 hybrids such as DH20 × Kate A-1 and DH6 × Altay 2000 with low anther response gave better results in terms of haploid embryo formation and regeneration. The means of fertile spike percentages and number of seeds per fertile spike were 26.75% and 9.83, respectively in developed green plants. As a result, bread wheat × sweet maize hybridization will be a good alternative to obtain a homozygous line in a short time in bread wheat genotypes with low anther response.

Effect of sucrose on calus induction and green plantlet regeneration in anther culture of Indica x Indica rice

Anther culture is an important biotechnological tool. By rice anther culture homozygous pure lines in the form of doubled haploid (DH) plants can be produced within a year as compared to the long inbreeding method, which might take 8-10 years. Application of anther culture technique in breeding and genetics research is limited due to the very low regeneration frequency of anthers of rice in general, and indica cultivars in particular. Therefore, the successful use of the technique depends on the adequate production of DH plants for selection and field evaluation. Effect of various concentrations of sucrose in callus induction media was investigated on callus induction and regeneration of green plantlets from anther culture of several F1s derived from indica x indica crosses. Cold pretreated anthers of five genotype of anther donor plants (F1s) at 5 °C for 8 days were cultured on N6 callus induction medium containing sucrose at a concentration of 6.0%, 6.5%, and 7.0%. Results revealed that all genotypes showed similar response to callus induction, but a significantly different responses to plant regeneration depended on sucrose level in the callus induction media. Two genotypes, i.e. IR78788 / Inpara 5 and Dendang/Inpari 30 only regenerated albino plantlets. In this experiment, 6.5% sucrose compared to two other sucrose treatments was suitable for inducing high frequency callus induction and high green plant regeneration in all genotypes. The high concentration of sucrose (7.0%) in the culture medium not only resulted in a decrease in the number and percentage of callus formation but also decreased the regeneration of green plantlets.

Androgenic studies in the production of haploids and doubled haploids in Capsicum spp.

Capsicum spp. is a horticultural crop of agronomic interest and is considered the fourth most important vegetable in the world. It is an important nutritional and medicinal source, and its production generates employment in the tropics. In this species, the genetic variability is wide and with great potential, which has been exploited to generate outstanding varieties. Breeding programs seek different alternatives to accelerate the production of improved varieties with desirable agronomic characteristics. These objectives can be achieved with the production of haploid and double haploid plants via androgenesis or gynogenesis, being androgenesis the approach most used for paprika cultures. The purpose of this review is to present the results of different researches in obtaining haploids and doubled haploids in cultivars of Capsicum spp. and its impact on the genetic improvement of this crop.

CRISPR/Cas9-Mediated Targeted Mutagenesis in Wheat Doubled Haploids.

CRISPR/Cas9-based genome editing technology has the potential to revolutionize agriculture, but many plant species and/or genotypes are recalcitrant to conventional transformation methods. Additionally, the long generation time of crop plants poses a significant obstacle to effective application of gene editing technology, as it takes a long time to produce modified homozygous genotypes. The haploid single-celled microspores are an attractive target for gene editing experiments, as they enable generation of homozygous doubled haploid mutants in one generation. Here, we describe optimized methods for genome editing of haploid wheat microspores and production of doubled haploid plants by microspore culture.

Proteases with caspase 3-like activity participate in cell death during stress-induced microspore embryogenesis of Brassica napus

Abstract Microspore embryogenesis is a model system of plant cell reprogramming, totipotency acquisition, stress response and embryogenesis initiation. This in vitro system constitutes an important biotechnological tool for haploid and doubled-haploid plant production, very useful for crop breeding. In this process, microspores (cells that produce pollen grains in planta) are reprogrammed toward embryogenesis by specific stress treatment, but many microspores die after the stress. The occurrence of cell death is a serious limiting problem that greatly reduces microspore embryogenesis yield. In animals, increasing evidence has revealed caspase proteolytic activities as essential executioners of programmed cell death (PCD) processes, however, less is known in plants. Although plant genomes do not contain caspase homologues, caspase-like proteolytic activities have been detected in many plant PCD processes. In the present study, we have analysed caspase 3-like activity and its involvement in stress-induced cell death during initial stages of microspore embryogenesis of Brassica napus. After stress treatment to induce embryogenesis, isolated microspore cultures showed high levels of cell death and caspase 3-like proteolytic activity was induced. Treatments with specific inhibitor of caspase 3-like activity reduced cell death and increased embryogenesis induction efficiency. Our findings indicate the involvement of proteases with caspase 3-like activity in the initiation and/or execution of cell death at early microspore embryogenesis in B. napus, giving new insights into the pathways of stress-induced cell death in plants and opening a new way to improve in vitro embryogenesis efficiency by using chemical modulators of cell death proteases.

Barley Anther Culture.

The production of doubled haploid (DH) barley plants through anther culture is a very useful yet simple in vitro technique. DH plants derive from divisions of haploid microspores that have undergone a developmental switch under the appropriate conditions. The successive divisions lead to the formation of an embryo or callus rather than the formation of mature pollen grains. Plants that regenerate from these embryos are often either haploid, in which case their chromosome set can be doubled by treatment with colchicine, or spontaneous double haploids. The efficiency of DH plant production is highly variable depending on the genotype of the source material. Despite this limitation, DH plants have been widely used in breeding and research programs. Compared to conventional approaches, breeding strategies that makes use of DH plants achieve a homozygous state, allowing transgene or mutation stabilization in the genome, within a considerably shorter time, thus accelerating workflow or reducing work volume.

Enhancement of microspore embryogenesis induction and plantlet regeneration of sweet pepper (Capsicum annuum L.) using putrescine and ascorbic acid.

Production of doubled haploid (DH) plants is an efficient tool in genetic and plant breeding programs; however, sweet pepper (Capsicum annuum L.) is recalcitrant to microspore embryogenesis and DH production. Trying to break the barrier of DH production, three independent experiments were conducted on microspore embryogenesis of sweet pepper. In the first experiment, the effect of cold (4°C) and heat (32°C) pretreatments were investigated on microspore embryogenesis of three genotypes of sweet pepper including "Inspiration F1," "Maratus F1," and "Magno F1" cultivars in a factorial design with three replications. Heat shock (32°C for 7days), applied to mannitol-starved anthers of "Inspiration F1," showed higher multinuclear microspore percent, number of multicellular structures, total embryos, cotyledonary embryos, and regenerants. In the second experiment, the effect of different concentrations of putrescine (0, 0.5, 1, 2, and 5mgl-1) was evaluated on microspore embryogenesis of the three aforementioned cultivars of sweet pepper. The highest mean number of multicellular structures, cotyledonary embryos, and regenerants were achieved by applying 0.5-1mgl-1 putrescine during the mannitol starvation and heat shock (32°C) treatments of isolated microspore culture of "Inspiration F1" cultivar. Significant decrease in microspore embryogenesis efficiency was observed when high levels of putrescine (2 and 5mgl-1) were used. Microspore embryogenesis was prevented completely at 5.0mgl-1 putrescine. In the third experiment, the effect of different concentrations of ascorbic acid (0, 20, 50, 100, and 200mgl-1) was investigated and the results showed that the application of ascorbic acid (20 and 50mgl-1) during mannitol starvation and heat shock treatment (32°C) caused remarkable improvement in the number of produced cotyledonary embryos and their regeneration ability compared to control treatment. However, the application of higher levels of ascorbic acid (100 and 200mgl-1) inhibited microspore cell divisions and embryogenesis. In conclusion, the results indicated that both putrescine and ascorbic acid have significant effect on microspore embryogenesis efficiency of sweet pepper when they are used in appropriate concentrations.

Production and conversion of haploid embryos in chickpea (Cicer arietinum L.) anther cultures using high 2,4-D and silver nitrate containing media

Due to recalcitrant nature of chickpea (Cicer arietinum L.) to androgenesis, the production of double haploid plants has been only reported by Grewal et al. (Plant Cell Rep 28:1289–1299, 2009) using some physical stresses such as anther centrifugation and electrical shock. In the present study, we successfully obtained haploid plants from cultured anthers of two chickpea cultivars, Bivanij and Arman, using high 2,4-D and silver nitrate containing media without applying of these time and labor consuming stresses. For induction of androgenesis, different concentrations of 2, 4-D (0, 2, 5 and 10 mg/l) and silver nitrate (0, 5, 10, 15, 25 and 50 mg/l) were used in embryo development medium. In Bivanij cultivar, anther induction medium containing 10 mg/l 2,4-D and 15 mg/l silver nitrate produced the highest number of embryos (0.188) and regenerated plants (0.1) per each cultured anther, while the highest frequencies of embryos (0.1) and regenerated plants (0.075 and 0.063) were obtained from Arman cultivar when 10 mg/l 2,4-D was combined with 15 and 50 mg/l silver nitrate in anther culture medium, respectively. In second part of this study, different cold (4 °C for 4 and 7 days) and heat (30 °C for 10 days, 32 °C for 2 days and 35 °C for 8 h) pretreatments were applied on cultured anthers of Bivanij cultivar. Incubation of cultured anthers at 32 °C for 2 days significantly enhanced the rate of embryo formation up to 0.222 embryos per each anther, while the highest number of regenerated plants/anther (0.0332) was obtained when cold treated anthers at 4 °C for 7 days incubated at 30 °C for 10 days. Taken together, these results provide a good basis for large-scale generation of DH plants in this important legume species.

Identification of BABY BOOM homolog in bread wheat

Modern breeding practice of small grain cereals necessitates the development of an efficient system for the large scale and reproducible production of the doubled haploid (DH) lines. It is believed that among the available DH generation techniques, only isolated microspore culture (IMC) can satisfy the demand of public and private breeding programs. Unfortunately, the IMC method is prone to several challenges that jeopardizes its large scale adoption. One of the approaches to limit the variation in DH plant production and increase the efficiency of the method is manipulation of embryogenesis-related genes. Here we set up a study to map BABY BOOM in a bread wheat genome. The gene is one of the morphogenic regulators of somatic embryogenesis in plants. To achieve this task, we used amino acid sequences of Zea mays BBM-like proteins. TaBBM homoeologs were mapped to chromosomes 6AL, 6BL and 6DL. Amino acid sequence analysis revealed the presence of two AP2 domains and bbm-1 motif in the A and D copies and only one AP2 domain and bbm-1 motif in the B copy. This, along with the absence of both gene expression and predictable TATA-box, suggests that TaBBM-gB is a pseudogene. The expression pattern of the identified A and D homoeologs was similar to that for the BBM-like genes in other species and presence of the transcript was detected in an embryogenic microspore population. Identification of the TaBBM homolog can have application in elevating the efficiency of DH production, tissue culture, plant transformation and genome editing for wheat improvement.

Adsorption of cell-penetrating peptide Tat2 and polycation luviquat FC-370 to triticale microspore exine

Microspores are the pre-gametophyte stage of pollen, and have proven to be a successful tissue culture material for the production of doubled haploid plants. Microspore culture has also been used as a platform for the production of transgenic plants. The use of cell-penetrating peptides (CPPs) as transfection agents in microspores has been previously demonstrated, but at low efficiencies. Here, the pH dependent adsorption of the cationic CPP Tat2 to the sporopollenin surface of the microspore (the exine) has been explored using electrophoretic light scattering (ELS). Furthermore the adsorption of a commercially available polycationic polymer; Luviquat FC-370 (polyquaternium D-16, PQ-D16) was similarly measured using ELS. It was found that a suspension media with a pH less than 7.0 showed an approximately ten fold decrease in the amount of Tat2 that was required before apparent surface neutralization. This data suggests that a change in the surface chemistry of the microspore occurs in acidic pH conditions, that modulates the binding affinity of the CPP Tat2 in a non-trivially complex manner.

Production of doubled haploid plants from anther cultures of borage (Borago officinalis L.) by the application of chemical and physical stress

Anther culture can be used as a powerful tool to produce doubled haploid (DH) lines in medicinal plants, thus accelerating breeding programs. In the particular case of borage (Borago officinalis L.), a method to produce DH plants has not been yet published. In this work we evaluated the effect of different culture media and of different chemical (colchicine and n-butanol) and physical stresses (centrifugation and electroporation) on androgenesis induction and plant regeneration in borage anther cultures. We found that the highest response can be obtained with culture medium containing B5 salts and NLN vitamins, the addition of 200 mg/l colchicine during 4 days, a pretreatment of anthers with 0.2% n-butanol for 5 h, or the application to anthers of single physical stresses (either centrifugation at 300×g or a 100 V electrical shock, but not combined). This is the first report on the production of DH plants in borage. Together, the results presented hereby can be used as a basic framework for large-scale generation of DH plants in this important medicinal species.

Anther culture as an effective tool in winter wheat (Triticum aestivum L.) breeding.

The aim of this study was to determine the effect of genotype and induction medium in anther culture of wheat (Triticum aestivum L.). Ten F1 winter wheat genotypes were tested in anther culture (AC) to compare the two most frequently applied induction media (W14mf and P4mf). Androgenesis was induced during the treatment of each tested genotypes and green plants were produced from them using both media. Based on statistical analysis, the genotypes significantly influenced (at the 0.001 probability level) the efficiency of AC (embryo-like structures (ELS), albinos, green plantlets and transplanted plantlets) and the media also had a significant effect on the number of ELS and albino plantlets. Both media can be used for AC in wheat doubled haploid (DH) plant production. The production of ELS and green plantlets was higher in P4mf medium (48.84 ELS/100 anthers, 4.82 green plantlets/100 anthers) than in W14mf medium (28.14 ELS/100 anthers, 4.59 green plantlets/100 anthers). However, the green plant regeneration efficiency of the microspore-derived structures was 16.9% when using W14mf medium, while this value was 9.6% in the case of ELS induced with P4mf medium. The application of W14mf medium thus proved to be time- and labour-saving medium in the large-scale production of DH wheat plants. In our experiments, 267 DH plants were produced for our winter wheat breeding program. The spontaneous rediploidization rate was 32.72%.

Comparison of different androgenesis protocols for doubled haploid plant production in ornamental pepper (Capsicum annuum L.)

Doubled haploid (DH) plant production plays an important role in pepper (Capsicum annuum L.) breeding for development of new varieties. However, information is lacking for DH plant production in pepper genotypes carrying ornamental value. The major goal of this study was to produce DH ornamental pepper lines having the mentioned value. Androgenic responses of 48 genotypes at the F2 or F3 generation were compared in each of three androgenesis protocols to determine the most effective method. Of the three protocols tested, anthers were placed on two different semisolid culture media and on a double-layer medium also called shed-microspore culture medium. The results revealed that the shed-microspore culture protocol was superior to both semisolid anther culture protocols. The average numbers of total and normal-looking embryos per bud of the most responsive genotype were 102.90 and 34.11, respectively. We report here, a total of 122 regenerated ornamental pepper plants with ornamental value; 63 DH, 52 haploid, and 7 mixoploid plants were produced in the present study. The spontaneous diploidization rate was 51.6% based on flow cytometry analyses. The results showed that the shed-microspore culture protocol could be used effectively for the development of DH lines in ornamental pepper breeding programs.

Increased Recovery of Green Doubled Haploid Plants from Barley Anther Culture

ABSTRACTAlbinism is a major limitation in the production of doubled haploid plants in cereals. As this trait is partly genetically controlled, the culture method has to be adapted to the plant genotype to reduce albinism. We have improved green plant recovery in the Northern barley (Hordeum vulgare L.) spring cultivar Mitja from 6 to 42% by altering the culture method and composition of the culture media. In a new three‐step protocol, the addition of casein hydrolysate to an intermediate regeneration medium gave the highest number of green plants. The shoot and root growth medium used for germinated embryos was superior to the standard media reported in the literature for root formation and shoot growth. Our modified method has merit for testing with other recalcitrant genotypes in anther and isolated microspore cultures.